Prostaglandin E Synthase Inhibitors and Methods for Utilizing the Same

ABSTRACT

Compounds and compositions are provided that can inhibit microsomal prostaglandin E synthase-1 (mPGES-1). The compounds and compositions can reduce inflammation in a subject, such as inflammation caused by an inflammation disorder or symptoms thereof. Pharmaceutical compositions comprising the compound are also provided. Furthermore, methods are provided for reducing inflammation and/or inhibiting mPGES-1. The methods can comprise administering an effective amount of the composition to a subject.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/355,739, filed Jun. 28, 2016, the entire disclosure of which isincorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to prostaglandin Esynthase (PGES) inhibitors, and in particular, microsomal PGES-1(mPGES-1) inhibitors. Embodiments of the presently-disclosed subjectmatter also relate to methods of utilizing mPGES-1 inhibitors to treatinflammatory disorders in a subject in need thereof.

BACKGROUND

Prostaglandin E₂ (PGE₂) is one of the most important prostanoids withdiverse biological activity.¹ The biosynthetic pathway of PGE₂ has beenwell characterized and involves three sequential enzymatic actions.² Thefirst step in this pathway, involves the release of arachidonic acid(AA) from the membrane, by the action of phospholipase A₂ (PLA₂).² Thisis followed by the conversion of AA to prostaglandin H₂ (PGH₂) by theaction of cyclooxygenase COX-1 or COX-2.² Finally, PGH₂ is converted toPGE₂ by the action of terminal prostaglandin E synthase (PGES) enzymes,³particularly microsomal PGES-1 (mPGES-1).⁴ It has been known thatmPGES-1 couples with COX-2⁵⁻⁶ and plays a key role in a number ofdisease conditions, including inflammation, arthritis, fever, pain,cancer, stroke, and bone disorders.⁷⁻¹³ Human mPGES-1 has beenrecognized as a promising target of next-generation therapeutics for theabove diseases.¹⁴

Currently available non-steroidal anti-inflammatory drugs (NSAIDs)inhibit either cyclooxygenase (COX)-1 or COX-2 or both.¹⁵ Theseinhibitors have several deleterious side effects including ulcers,bleeding within the gastrointestinal tract, or increased risk ofcardiovascular events.¹⁶ The withdrawal of rofecoxib (Vioxx) due to sideeffects further highlights the need to develop improved, saferanti-inflammatory drugs.¹⁵ The COX inhibitors prevent the production ofall prostaglandins downstream of PGH₂, which results in a lot ofproblems. For example, blocking the production of prostaglandin-I₂(PGI₂) has been reported to play a role in cardiovascular events.¹⁷Unlike COX inhibition, inhibition of terminal mPGES-1 will only blockthe production of PGE₂ without affecting the normal production of otherprostaglandins including PGI₂. Reported knock-out studies identifiedmPGES-1 as a central switch in pyresis.¹⁸ The mPGES-1 knock-out studiesalso revealed a decrease in inflammatory response in a collagen-inducedarthritis model.¹⁹ In contrast to COX-2, mPGES-1-deficient mice werereported to be viable, fertile and have normal phenotype.¹⁹ Ischemicstroke induced in mPGES-1 null mice was reported to show significantreduction in the infarct size and volume.^(10, 14) Thus, mPGES-1inhibitors are expected to retain the anti-inflammatory effect as COXinhibitors without the side effects of COX inhibitors.

Although mPGES-1 inhibitors are expected to be potentially valuabletherapeutic agents, few inhibitors of mPGES-1 were identified inexperimental screening efforts. The COX-2 inhibitor NS-398,5-Lipoxygenase activating protein (FLAP) inhibitor MK-886, and theactive metabolite of another NSAID sulindac, were found to inhibitmPGES-1 with an IC₅₀ of 20, 1.6, and 80 μM, respectively.^(20-21,22)Leukotriene C4 was reported to inhibit mPGES-1 with micromolar IC₅₀,probably by competing with glutathione (GSH).²⁰ In addition to smallmolecules,²³ several polyunsaturated fatty acids and stable analogs ofPGE₂ were reported to inhibit mPGES-1.²⁴ Riendeau²² recently reported aseries of mPGES-1 inhibitors synthesized based on the scaffold of MK-886(FLAP inhibitor). Unfortunately, all of these inhibitors are notsufficiently potent against mPGES-1 in the tested living cells.

Thus, there remains a need for novel compounds that more potentlyinhibit mPGES-1. There also remains a need for methods of treatinginflammatory disorders that do not have the problems discussed above.However, known mPGES-1 inhibitors are not sufficiently potent, and knownanti-inflammatory agents are associated with many adverse side effects,such as ulcers and gastrointestinal bleeding. Hence, novel compoundsthat more potently inhibit mPGES-1 activity and are thereby able totreat inflammatory disorders are highly desired.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof; wherein R¹ is selectedfrom the group consisting of H, halide, Me, OMe, OEt, NO₂, OH, and,together with the ring to which it is attached, a bicyclic ring system;wherein R² is alkyl; wherein R³ is selected from the group consisting ofH and Me; and wherein X is selected from the group consisting of O or S.In one embodiment, R¹ is selected from the group consisting of: H, Cl,Br, I, Me, OMe, OEt, NO₂, OH, and, taken together with the ring to whichit is attached, a bicyclic ring system. In another embodiment, R² isselected from the group consisting of:

In a further embodiment, the compound includes the formula selected fromthe group consisting of:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R¹ is selectedfrom the group consisting of H, an alkyl, an alkyl halide, an ether, anda carboxylic acid; wherein R² is selected from the group consisting ofH, a halide, an alkyne, and an aromatic; and wherein R³ is selected fromthe group consisting of H, a carboxyl, a carboxylic acid, and an alkyl.In one embodiment, R¹ is selected from the group consisting of:

H,

In another embodiment, IV is selected from the group consisting of:

H,

In a further embodiment, R³ is selected from the group consisting of:

H,

In some embodiments, the compound includes the formula selected from thegroup consisting of:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected fromthe group consisting of an alkyl and an alkoxy; and wherein n is from 1to 6. In one embodiment, R is selected from the group consisting of:

In another embodiment, the compound has the formula selected from thegroup consisting of:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is a substitutedphenyl; and wherein X is O. In one embodiment, the compound is of theformula:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected fromthe group consisting of an aliphatic side chain and an alkyl; wherein Xis selected from the group consisting of H, NO₂, Br, and OMe; andwherein R′ and R″ are independently selected from the group consistingof CN, COOH, COOEt, CONH₂, and NO₂ In one embodiment, the compound is ofthe formula:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R¹ is an alkyl;wherein each R² is independently selected from the group consisting of Hand an alkyl; wherein X is selected from the group consisting of H and ahalogen; and wherein Y is selected from the group consisting of S and O.In one embodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes acompound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected fromthe group consisting of an aliphatic side chain and an alkyl; wherein Xis selected from the group consisting of H and Cl; wherein Y is CN;wherein Z is selected from the group consisting of CN, COOH, and,together with Y, a heterocyclic group of the formula:

wherein R¹ is selected from the group consisting of O and S; and whereinR² is selected from the group consisting of H and CH₂COOH. In oneembodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes apharmaceutical composition comprising one of the compounds disclosedherein and a pharmaceutically-acceptable carrier. In one embodiment, thepharmaceutical composition further comprises a second compound orcomposition having mPGES-1 inhibition activity, having anti-inflammatoryactivity, being useful for treatment of an inflammation disorder, beinguseful for treatment of symptoms associated inflammation and/or aninflammation disorder, or combinations thereof.

In some embodiments, the presently-disclosed subject matter includes akit comprising one of the compounds disclosed herein and a device usefulfor administration of the compound. In one embodiment, the kit furthercomprises a second compound or composition, or a treatment device havingmPGES-1 inhibition activity, anti-inflammatory activity, being usefulfor treatment of an inflammation disorder, and/or being useful fortreatment of symptoms associated inflammation and/or an inflammationdisorder.

In some embodiments, the presently-disclosed subject matter includes amethod of reducing inflammation in a subject, comprising administeringto the subject an effective amount of one of the compound disclosedherein. In one embodiment, the subject includes an inflammation disorderor symptoms thereof. In another embodiment, the inflammation disorder isselected from the group consisting of inflammation, arthritis, fever,pain, cancer, stroke, bone disorders, and combinations thereof. In afurther embodiment, the compound inhibits microsomal prostaglandin Esynthase-1 (mPGES-1).

In some embodiments, the presently-disclosed subject matter includes amethod of reducing inflammation in a subject, comprising administeringto the subject an effective amount of a compound selected from the groupconsisting of:

In one embodiment, the subject includes an inflammation disorder orsymptoms thereof. In another embodiment, the inflammation disorder isselected from the group consisting of inflammation, arthritis, fever,pain, cancer, stroke, bone disorders, and combinations thereof. In afurther embodiment, the compound inhibits microsomal prostaglandin Esynthase-1 (mPGES-1).

Further features and advantages of the presently-disclosed subjectmatter will become evident to those of ordinary skill in the art after astudy of the description, figures, and non-limiting examples in thisdocument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic view of the general synthesis of compoundshaving the structure of formula I, according to an embodiment of thedisclosure. Reagents and conditions: (a) 50% KOH aq., DCM, 0° C.˜rt; (b)K₂CO₃ (2.0 equiv.), Acetone, reflux; (c) K₂CO₃ (2.0 equiv.), DMF, 80°C.; (d) EtOH/H₂O (4:1, v/v), reflux.

FIG. 2 shows a schematic view of the synthesis of a specific compoundhaving the structure of formula I, according to an embodiment of thedisclosure. Reagents and conditions: (a) 1) NaBH₄ (1.25 equiv.), MeOH,0° C.˜rt, 2) 1 M HCl solution, rt.

FIG. 3 shows a schematic view of the general synthesis of compoundshaving the structure of formula II, according to an embodiment of thedisclosure. Reagents and conditions: (a) Triphenylphosphine (1.20equiv.), DIAD (1.20 equiv.), THF, 0° C.˜rt; (b) acetone, reflux; (c)TFA/DCM (1:1, v/v), rt; (d) K₂CO₃ (2.0 equiv.), DMF, 80° C.; (e)Pd(dppf)Cl₂.CH₂Cl₂ (0.03 equiv.), NaHCO₃ (2.50 equiv.), DME/H₂O, reflux,N₂ atmosphere; (0 ammonium acetate (2.00 equiv.), glacial acetic acid,reflux.

FIG. 4 shows a schematic view of the synthesis of a specific compoundhaving the structure of formula II, according to an embodiment of thedisclosure. Reagents and conditions: (a) Triphenylphosphine (1.20equiv.), DIAD (1.20 equiv.), THF, 0° C.˜rt; (b) K₂CO₃ (2.0 equiv.), DMF,80° C.; (c) TFA/DCM (1:1, v/v), rt; (d) NH₄OAc (2.00 equiv.), glacialAcOH, reflux.

FIG. 5 shows a schematic view of the general synthesis of compoundshaving the structure of formula III, according to an embodiment of thedisclosure. Reagents and conditions: (a) Glacial AcOH, reflux; (b) HBTU(1.10 equiv.), DIPEA (3.30 equiv.), DMF, 0° C.˜rt; (c)Triphenylphosphine (1.20 equiv.), DIAD (1.20 equiv.), THF, 0° C.˜rt; (d)H₂, Pd/C (10% w/w), THF/MeOH (4:1 v/v), rt.

FIG. 6 shows a schematic view of the general synthesis of compoundshaving the structure of formula IV, according to an embodiment of thedisclosure. Reagents and conditions: (a) Triphenylphosphine (1.20equiv.), DIAD (1.20 equiv.), THF, 0° C.˜rt; (b) NH₄OAc (2.00 equiv.),glacial AcOH, reflux; (c) K₂CO₃ (2.0 equiv.), DMF, 80° C.

FIG. 7 shows a schematic view of the general synthesis of compoundshaving the structure of formula V, according to an embodiment of thedisclosure. Reagents and conditions: (a) K₂CO₃ (2.00 equiv.), DMF, 80°C.; (b) Malononitrile, NH₄OAc (2.00 equiv.), AcOH, reflux.

FIG. 8 shows a schematic view of the general synthesis of compoundshaving the structure of formula VI, according to an embodiment of thedisclosure. Reagents and conditions: (a) K₂CO₃ (2.00 equiv.), DMF, 80°C.; (b) 5% glacial AcOH in EtOH, reflux; (c) POCl₃ (4.00 equiv.), DMF,0° C.˜60° C.; (d) EtOH/H₂O (4:1, v/v), reflux.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.To avoid excessive repetition, this Description does not list or suggestall possible combinations of such features.

The presently-disclosed subject matter includes a compound having astructure represented by the formula I:

or pharmaceutically acceptable salts thereof, wherein R¹ includes H,halide, Me, OMe, OEt, NO₂, OH, or taken together with the ring to whichit is attached, a bicyclic ring system as in the formula:

R² includes an alkyl; R³ includes H or Me; and X includes O or S.

In some embodiments of the compound having the structure of formula I,R¹ includes H, Cl, Br, I, Me, OMe, OEt, NO₂, OH, or taken together withthe ring to which it is attached, a bicyclic ring system.

In some embodiments of the compound having the structure of formula I,R² includes:

In some embodiments of the compound of formula I, the compound has thestructure selected from the group consisting of:

In some embodiments of the compound of formula I, the compound has thestructure of:

The presently-disclosed subject matter includes a compound having astructure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R¹ includes H, analkyl, an alkyl halide, an ether, or a carboxylic acid; R² includes H, ahalide, an alkyne, or an aromatic; and R³ includes H, a carboxyl (e.g.,CO₂H), a carboxylic acid (e.g., CH₂CO₂H), or an alkyl.

In some embodiments of the compound having the structure of formula II,R¹ is selected from the group consisting of:

H,

In some embodiments of the compound having the structure of formula II,R² is selected from the group consisting of:

H,

In some embodiments of the compound having the structure of formula II,R³ is selected from the group consisting of:

H,

In some embodiments of the compound having the structure of formula II,the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter includes a compound having astructure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R is an alkyl oralkoxy; and n is 1, 2, 3, 4, 5, or 6.

In some embodiments of the compound having the structure of formula III,R is selected from the group consisting of:

In some embodiments of the compound having the structure of formula III,the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter includes a compound having astructure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected fromthe group consisting of substituted phenyl; and X is O.

In some embodiments, the compound of formula IV has the structure of

The presently-disclosed subject matter includes a compound having astructure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula V, Rincludes an aliphatic side chain or an alkyl; X is selected from thegroup consisting of H, NO₂, Br, or OMe; and R′ and R″ are independentlyselected from the group consisting of CN, COOH, COOEt, CONH₂, and NO₂.

In some embodiments of the compound having the structure of formula V,the compound is selected from the group consisting of:

The presently-disclosed subject matter includes a compound having astructure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula VI,X includes H or a halogen such as Cl; IV includes an alkyl; each R²independently includes H or an alkyl; and Y includes S or O.

In some embodiments of the compound having the structure of formula VI,the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter further includes a compoundhaving a structure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula VII,X includes H or Cl; Y includes CN; Z includes CN or COOH; and R includesan aliphatic side chain or an alkyl. In some embodiments of the compoundhaving the structure of formula VII, Y and Z together form aheterocyclic group, such as, but not limited to, a five memberedheterocyclic group. In one embodiment, for example, Y and Z togetherform a thiazolidine group having the structure:

wherein R¹ includes O or S; and R² includes H or CH₂COOH.

In some embodiments of the compound having the structure of formula VII,the compound has the structure selected from the group consisting of:

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids. When thecompound of the present invention is acidic, its corresponding salt canbe conveniently prepared from pharmaceutically acceptable non-toxicbases, including inorganic bases and organic bases. Salts derived fromsuch inorganic bases include aluminum, ammonium, calcium, copper (-icand -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and-ous), potassium, sodium, zinc and the like salts. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines, as wellas cyclic amines and substituted amines such as naturally occurring andsynthesized substituted amines. Other pharmaceutically acceptableorganic non-toxic bases from which salts can be formed include ionexchange resins such as, for example, arginine, betaine, caffeine,choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

The term “alkyl” refers to alkyl groups with the general formulaC_(n)H_(2n+1), where n=about 1 to about 18 or more. The groups can bestraight-chained or branched. Alkyl, when used herein, also comprise“lower alkyls,” which refer to alkyl groups with the general formulaC_(n)H_(2n+1), where n=1 to about 6. In some embodiments, n=1 to about3. Examples include methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, t-butyl, isobutyl, n-pentyl, isopentyl, neopentyl, n-hexyl,and the like. The alkyl group can be substituted or unsubstituted. Forexample, the alkyl group can be substituted with one or more groupsincluding, but not limited to, optionally substituted alkyl, cycloalkyl,alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, orthiol, as described herein.

In this regard, the term alkyl is inclusive of “cycloalkyl,” whichrefers to a non-aromatic carbon-based rings composed of at least threecarbon atoms, such as cyclopropyl, cyclohexyl, and the like. Like otheralkyls, cycloalkyls can be substituted or unsubstituted. The substitutedmoieties can be specifically identified herein; for example, aparticular substituted cycloalkyl can be referred to as an“alkylcycloalkyl.” Again, the practice of using a general term, such as“cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is notmeant to imply that the general term does not also include the specificterm.

The term “fluorocarbon” refers to compounds that comprise carbon andfluoride bonded together. Fluorocarbons can comprise any type of bondand may be fluoroalkyl, fluoroalkene, or the like. Examples offluorocarbons include CF₄, C₂F₆, C₂F₄, and the like.

The term “aryl,” refers to an aromatic group containing ring carbonatoms and having about 5 to about 14 ring carbon atoms and up to a totalof about 18 ring or pendant carbon atoms. Examples include, but are notlimited to, phenyl, biphenyl, naphthalene, α-naphthyl, β-naphthyl,tolyl, xylyl, benzene, phenoxybenzene, and the like. The term “aryl”also includes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Likewise, the term “non-heteroaryl,” whichis also included in the term “aryl,” defines a group that contains anaromatic group that does not contain a heteroatom. The aryl group can besubstituted or unsubstituted. The term “biaryl” is a specific type ofaryl group and is included in the definition of “aryl.” Biaryl refers totwo aryl groups that are bound together via a fused ring structure, asin naphthalene, or are attached via one or more carbon-carbon bonds, asin biphenyl.

As described above, each of the groups mentioned herein, including thegroups defined above, could be substituted or unsubstituted. Forexample, “alkyl” can include substituted alkyl, substituted withhydroxyl, heteroatoms, or lower alkyl groups. As a further example,“aryl” can include substituted aryl, substituted with alkyl, cycloalkyl,amino, nitro, thiol, or the like.

Compounds described herein can potentially give rise to cis/trans (E/Z)isomers, as well as other conformational isomers. Unless stated to thecontrary, the presently-disclosed subject matter includes all suchpossible isomers, as well as mixtures of such isomers. Unless stated tothe contrary, a formula with chemical bonds shown only as solid linesand not as wedges or dashed lines contemplates each possible isomer,e.g., each enantiomer and diastereomer, and a mixture of isomers, suchas a racemic or scalemic mixture. Compounds described herein can containone or more asymmetric centers and, thus, potentially give rise todiastereomers and optical isomers. Unless stated to the contrary, thepresent compounds all such possible diastereomers as well as theirracemic mixtures, their substantially pure resolved enantiomers, allpossible geometric isomers, and pharmaceutically acceptable saltsthereof. Mixtures of stereoisomers, as well as isolated specificstereoisomers, are also included. During the course of the syntheticprocedures used to prepare such compounds, or in using racemization orepimerization procedures known to those skilled in the art, the productsof such procedures can be a mixture of stereoisomers.

The presently-disclosed subject matter further includes pharmaceuticalcompositions of the compounds as disclosed herein, and further includesa pharmaceutically-acceptable carrier. In this regard, the term“pharmaceutically acceptable carrier” refers to sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose.

Suitable formulations include aqueous and non-aqueous sterile injectionsolutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

The compositions can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compounds can also be formulated as a preparation for implantationor injection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (e.g., as a sparingly soluble salt).

The compounds can also be formulated in rectal compositions (e.g.,suppositories or retention enemas containing conventional suppositorybases such as cocoa butter or other glycerides), creams or lotions, ortransdermal patches.

The compounds disclosed herein have utility as PGES inhibitors, and inparticular, mPGES-1. In this regard, the compounds and pharmaceuticalcompositions of the presently disclosed subject matter haveanti-inflammatory utilities. In this regard, in some embodiments, thepharmaceutical compositions of the presently-disclosed subject matterfurther include a second compound having PGES inhibition activity,having anti-inflammatory activity, being useful for treatment of aninflammation disorder, and/or being useful for treatment of symptomsassociated inflammation and/or an inflammation disorder.

The presently-disclosed subject matter further includes kits. In someembodiments, a kit can include a compound or pharmaceutical compositionas described herein, packaged together with a second compound,composition, or treatment device having PGES inhibition activity, havinganti-inflammatory activity, being useful for treatment of aninflammation disorder, and/or being useful for treatment of symptomsassociated inflammation and/or an inflammation disorder. By way ofproviding non-limiting examples of treatment devices that could beincluded in a kit of the presently-disclosed subject matter,inflammation can be treated in some cases with application of a devicethat changes temperature at a site of interest, e.g., a cooling pack ora heating pack.

In some embodiments, a kit can include a compound or pharmaceuticalcomposition as described herein, packaged together with a device usefulfor administration of the compound or composition. As will be recognizedby those or ordinary skill in the art, the appropriate administrationaiding device will depend on the formulation of the compound orcomposition that is selected and/or the desired administration site. Forexample, if the formulation of the compound or composition isappropriate for injection in a subject, the device could be a syringe.For another example, if the desired administration site is cell culturemedia, the device could be a sterile pipette.

The presently-disclosed subject matter further includes methods ofinhibiting mPGES. In some embodiments, the method can include contactingany of the compounds or compositions described herein with mPGES-1,thereby forming a complex with the compound and mPGES-1. In someembodiments, the method can include administering an effective amount ofa compound or pharmaceutical composition, as described herein, includingbut not limited to the compounds set forth herein, and compositionsthereof.

As will be recognized by one of ordinary skill in the art, the term“inhibiting” or “inhibition” does not refer to the ability to completelyinactivate all target biological activity in all cases. Rather, theskilled artisan will understand that the term “inhibiting” refers todecreasing biological activity of a target, such as a prostaglandin Esynthase, such as can occur with a ligand binding site of the target isblocked, or when a non-native complex with the target is formed. Suchdecrease in biological activity can be determined relative to a control,wherein an inhibitor is not administered and/or placed in contact withthe target. For example, in some embodiments, a decrease in activityrelative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%decrease. The term “inhibitor” refers to a compound of composition thatinactivates or decreases the biological activity of a target, such as aprostaglandin E synthase.

Without being bound by theory or mechanism, in some embodiments thecompounds disclosed herein inhibit mPGES-1 by blocking its interactionwith the PGH₂, COX-2, or other substrates. Thus, the presently-disclosedsubject matter also includes methods that find utility from the blockingmPGES-1 interaction with PGH₂, COX-2, or other substrates. In thisregard, the presently-disclosed subject matter includes methods ofreducing and/or inhibiting inflammation, and methods of treating aninflammation disorder, and/or symptoms associated inflammation and/or aninflammation disorder. Such methods can include administering aneffective amount of a compound of pharmaceutical composition asdescribed herein to a subject. Non-limiting examples of inflammationdisorders include inflammation, arthritis, fever, pain, cancer, stroke,and bone disorders

In some embodiments of a method of treating an inflammation disorder orsymptoms thereof in a subject in need thereof, the method includesadministering to the subject an effective amount of a compound,including any of the compounds described above. In some embodiments thecompound inhibits prostaglandin E synthase (PGES), and in particular,some embodiments inhibit microsomal PGES-1 (mPGES-1). Thus, someembodiments include a method for inhibiting mPGES-1, comprisingadministering to a subject an effect amount of a compound, including anyof the compounds described above.

The terms “treatment” or “treating” refer to the medical management of apatient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The terms “subject” or “subject in need thereof” refer to a target ofadministration, which optionally displays symptoms related to aparticular disease, pathological condition, disorder, or the like. Thesubject of the herein disclosed methods can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. A patient refers to a subject afflicted with a disease ordisorder. The term “patient” includes human and veterinary subjects.

In some embodiments, compounds disclosed herein that are mPGES-1inhibitors are potent against both human and mouse mPGES-1 enzymes.

The term “administering” refers to any method of providing apharmaceutical preparation to a subject. Such methods are well known tothose skilled in the art and include, but are not limited to, oraladministration, transdermal administration, administration byinhalation, nasal administration, topical administration, intravaginaladministration, ophthalmic administration, intraaural administration,intracerebral administration, rectal administration, and parenteraladministration, including injectable such as intravenous administration,intra-arterial administration, intramuscular administration, andsubcutaneous administration. Administration can be continuous orintermittent. In various aspects, a preparation can be administeredtherapeutically; that is, administered to treat an existing disease orcondition. In further various aspects, a preparation can be administeredprophylactically; that is, administered for prevention of a disease orcondition.

The term “effective amount” refers to an amount that is sufficient toachieve the desired result or to have an effect on an undesiredcondition. For example, a “therapeutically effective amount” refers toan amount that is sufficient to achieve the desired therapeutic resultor to have an effect on undesired symptoms, but is generallyinsufficient to cause adverse side affects. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

The presently-disclosed subject matter further includes methods forselecting and synthesizing embodiments of the present invention canutilize structure-based virtual screening to identify small-moleculeinhibitors from a large drug-like database. In some embodiments a largedatabase of lead compounds can be virtually screened to retrieveputative mPGES-1 inhibitors. From that screening, essential amino acidsinvolved in antagonist recognition can be identified and a primarytopographical interaction model can be made to guide subsequent virtualscreening processes. Without being bound by theory or mechanism, aninhibitor's binding pocket of the mPGES-1 protein can overlap with boththe binding site of the PGH₂ substrate and GSH cofactor in mPGES-1protein.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. Some examples areprophetic. Some of the following examples may include compilations ofdata that are representative of data gathered at various times duringthe course of development and experimentation related to thepresently-disclosed subject matter.

EXAMPLES Example 1 Synthetic Protocol Compounds of Formula I

The synthesis of BAR series (compounds of Formula I) can be generallydescribed by the schemes illustrated in FIG. 1 and FIG. 2 of thisExample. (for BAR042-044).

The substituted hydroxybenzaldehyde or hydroxy naphthaldehyde wastreated with alcohol tosylate or alkyl bromide in the presence ofpotassium carbonate as acid capturer.^(25,26) The forming aldehydeintermediate was usually pure enough after aqueous work-up and removalof solvents which could be used for the subsequent step without furtherpurification. However, analytical samples could be obtained by flashchromatography using a mixture of hexanes and ethyl acetate as eluent.The final product, substituted benzylidenebarbituric acid derivativeswere obtained by the condensation of the aldehyde intermediate andbarbituric acid (or 1,3-dimethylbarbituric acid, 2-thiobarbituric acid)in reflux ethanol/water (4:1, v/v).^(27,28) The precipitate formed waswashed with hot water and ethanol, and dried under vacuum to form theanalytical pure sample.

I-42˜I-44 were synthesized by the reduction of benzylidene double bondof I-02, I-03, and I-08, using sodium borohydride in methanol asreducing agent solution.²⁹

Example 2 Synthetic Protocol of the Compounds of Formula II

Commercially available isatin (or 5-iodoisatin) and2,4-thiazolidinedione were used as starting materials to construct thebuilding blocks of substituted isatin and 2,4-thiazolidinedione N-aceticacid, respectively. After treatment with potassium hydroxide in hotethanol, the potassium salt of 2,4-thiazolidinedione was precipitatedout for the N-substitution by tert-butyl bromoacetate. The removal oftert-butyl ester in TFA/DCM (1:1, v/v) at room temperature led to theformation of important building block 2,4-thiazolidinedione N-aceticacid.³² N-substituted isation was prepared by potassium carbonatepromoted reaction between isatin and alkyl bromide (or alcohol tosylateif the bromide was not commercially available).³³ While for1,5-disubstituted isatin, N-substitution on 5-iodoisatin by4-chlorobenzyl bromide in the presence of potassium carbonate wasfollowed by the Suzuki cross-coupling reaction with aryl boronic acid(ArB(OH)₂) using[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane (0) as catalyst and sodium bicarbonate as base activatorin refluxing dimethoxyethane (DME) and distilled water (DME/H₂O 4:1)under the protection of nitrogen gas.^(34,35) The final product wasobtained as red to brown powders by the Knovenagal-type condensation ofthe isatin-based building block with 2,4-thiazolidinedione N-acetic acidin the presence of ammonium acetate in refluxing glacial acetic acid, asdescribed in FIG. 3.

Some of the compounds in this series were designed by switching thepositions of hydrophilic and hydrophobic groups, as Cy4TZISA, whoseacetic acid group occupied N-position of isatin and aliphatic grouplinked to 2,4-thiazolidinedione moiety. These compounds were readilysynthesized following similar protocol as described previously, as shownin FIG. 4.

Example 3 Synthetic Protocol of Compounds of Formulae III and IV(Maleimide Derivatives and Substituted Dinitrobenzene Derivatives)

Maleimide derivatives were synthesized via the condensation of4-maleimidobutyric acid or 6-maleimidohexanoic acid³⁶ with arylamines,³⁷ as shown in FIG. 5.

In the synthesis of dinitrobenzene derivatized potent inhibitors, theoxygen on phenyl derivative nucleophilically substituted one of thefluorine atom on 1,2-difluoro-4,5-dinitrobenzene using potassiumcarbonate as acid capturer, as shown in FIG. 6.³⁸

Example 4 Synthetic Protocol of Compounds of Formula V

The compounds of Formula V were prepared following a two-stepprotocol.³⁹⁻⁴² O-Substitution of substituted 4-hydroxybenzaldehydeafforded the aldehyde intermediate and the latter was coupled withmalononitrile, 2-cyanoacetic acid or 2-cyanoacetamide. An example of thesynthesis of V-04 is illustrated in FIG. 7.

Example 5 Synthetic Protocol of Compounds of Formulae VI and VII

The compounds of Formula VI were synthesized according to a multi-stepprotocol. 4-Alkyloxyacetophenone, obtained from the reaction of4-hydroxyacetophenone and alkyl bromide, or acetophenone was condensedwith 4-chlorophenylhydrazine in reflux ethanol containing 5% glacialacetic acid. The ethylidene hydrazine was formed as precipitate at roomtemperature and filtered off. The next step was Vilsmeier-Haack-Arnoldring closing formylation, by treating with POCl₃/DMF. The produced1H-pyrazole-4-carbaldehyde intermediate was coupled with barbituric acidor 2-thiobarbituric acid in refluxing EtOH/H₂O (4:1) to afford the finalproduct. The synthetic protocol for compounds with Formula VII followedsimilar strategy as those with formula VI, except the final step whichwas the coupling with 2,4-thiazolidinedione derivatives. An example ofthe synthesis of VI-01 is depicted in FIG. 8.

Example 6 Characterization of Inhibition In Vitro

Studies were conducted to characterize the inhibitory activity againstrecombinant mPGES-1 of the compounds synthesized in accordance withExamples 1-4, and disclosed herein.

Briefly, FreeStyle 293-F cells were cultured following manufacturer'smanual in FreeStyle 293 expression medium on orbit rotate shaker in 8%CO₂ incubator at 37° C. Cells were transfected with 1.5 μg/mL ofmPGES-1/pcDNA3 construct using FreeStyle Max reagent at a cell densityof 1×10⁶ for 2 days. Transfected cells were collected, washed, andsonicated in TSES buffer (15 mM Tris-HCl, pH 8.0 plus 0.25 M sucrose,0.1 mM EDTA and 1 mM DTT) on ice. The broken cells were firstcentrifuged at 12,500×g for 10 min. The supernatant was furthercentrifuged at 105,000×g for 1 hr at 4° C. The pellet was washed andhomogenized in PBS buffer. The crude microsomal mPGES-1 preparation wasaliquoted and stored at −80° C. The crude protein concentration was 8mg/mL.

The enzyme activity assays were performed on ice in 1.5 ml microfugetubes by using the expressed mPGES-1. The reaction mixture contained:0.2 M Na₂HPO₄/NaH₂PO₄, pH 7.2, 10 μL; 0.1 M GSH, 2.5 μL; dilutedmicrosomal enzyme (80 μg/mL), 1 μL; PGH₂ (0.31 mM in DMF), 5 μL; 1 μl,inhibitor; and H₂O in a final reaction volume of 100 μL. PGH₂ was storedin dry ice and used to initiate the reaction.

Compounds were incubated with the enzyme for 15 min at room temperaturebefore the addition of cold PGH₂ (1 μM final) to initiate the enzymereaction. After 30 s, 10 μL of SnCl₂ (40 mg/mL) in ethanol was added tostop the reaction. The nonenzymatic conversion of PGH₂ to PGE₂ wasperformed in the same buffer devoid of enzyme. The reaction mixture wasplaced on ice until PGE₂ production was determined by the PGE₂ enzymeimmunoassay as described earlier. IC₅₀ values of the inhibitors werecalculated by using the GraphPad Prism 4.0 program. The results are setforth in the tables provided in Examples 7-10.

Example 7 Characterization of Inhibition of Compounds of Formula I

TABLE 1 IC₅₀ ^(a) against mPGES-1 (nM) Compound Structure Human enzymeMouse enzyme I-01

 622 ± 121  7079 ± 627 I-02

 33 ± 3  157 ± 31 I-03

 45 ± 8  917 ± 321 I-04

 82 ± 10 n.d.^(b) (25)^(c) I-05

 116 ± 17  2900 ± 293 I-06

 121 ± 20  1458 ± 209 I-07

 186 ± 26  2409 ± 339 I-08

 67 ± 20  698 ± 97 I-09

 22 ± 7  360 ± 56 I-10

 69 ± 16  292 ± 47 I-11

 54 ± 12  359 ± 50 I-12

 152 ± 53  727 ± 109 I-13

 87 ± 27 n.d. (28) I-14

 96 ± 14 n.d. (38) I-15

 135 ± 18 12078 ± 1963 I-16

 154 ± 18  7039 ± 1853 I-17

 171 ± 34  3699 ± 562 I-18

 272 ± 30 n.d. (8) I-19

 427 ± 55 n.d. (24) I-20

 561 ± 55 n.d. (15) I-21

 28 ± 3  415 ± 120 I-22

 20 ± 4  239 ± 72 I-23

 53 ± 14  9013 ± 1044 I-24

 110 ± 21  979 ± 84 I-25

 104 ± 25  336 ± 43 I-26

 156 ± 30  373 ± 51 I-27

 240 ± 20 n.d. (29) I-28

 127 ± 14 n.d. (30) I-29

 188 ± 21 n.d. (41) I-30

 78 ± 13  3231 ± 460 I-31

 112 ± 13  1444 ± 222 I-32

 73 ± 12  428 ± 58 I-33

 73 ± 10  2788 ± 525 I-34

 349 ± 40  8126 ± 1012 I-35

 337 ± 34 n.d. (17) I-36

 365 ± 59 n.d. (19) I-37

 517 ± 68  2395 ± 425 I-38

1114 ± 104 n.d. (42) I-39

 460 ± 64  6306 ± 1136 I-40

 232 ± 54  734 ± 119 I-41

 188 ± 43  1303 ± 163 I-42

 133 ± 20  1333 ± 151 I-43

 97 ± 13  1092 ± 211 I-44

 136 ± 23  3354 ± 560 ^(a)Data are expressed as means ±SD of singledeterminations obtained in triplicate. ^(b)n.d. = not detected. ^(c)The% inhibition of the compound at a concentration of 10 μM against mPGES-1(IC₅₀ values were determined if the compounds resulted in 50% or higherinhibition).

Example 8 Characterization of Inhibition of Compounds of Formula II

TABLE 2 IC₅₀ ^(a) against mPGES-1 (nM) Compound Structure Human enzymeMouse enzyme II-01

 817 ± 79 n.d.^(b) (10)^(c) II-02

 614 ± 63 n.d. (45) II-03

 348 ± 44 n.d. (62) II-04

2403 ± 132  747 ± 163 II-05

 963 ± 87 n.d. (61) II-06

1269 ± 104  2728 ± 422 II-07

 494 ± 32 n.d. (39) II-08

 253 ± 30  1518 ± 317 II-09

 499 ± 108  947 ± 183 II-10

 805 ± 100 n.d. (41) II-11

1681 ± 168  1023 ± 131 II-12

1661 ± 168 n.d. (35) II-13

1073 ± 116 n.d. (57) II-14

 324 ± 44 n.d. (67) II-15

 314 ± 44  2676 ± 302 II-16

 199 ± 32  1398 ± 217 II-17

 217 ± 30  935 ± 135 II-18

 221 ± 47  394 ± 58 II-19

 154 ± 20  1020 ± 162 II-20

 531 ± 90  244 ± 31 II-21

 91 ± 23  1960 ± 348 II-22

 25 ± 5  685 ± 406 II-23

8023 ± 1050 n.d. (63) II-24

 32 ± 6  777 ± 364 II-25

 16 ± 4  1222 ± 430 II-26

 13 ± 3  1130 ± 244 II-27

 54 ± 14 13236 ± 5018 II-28

1393 ± 273 n.d. (0) II-29

1263 ± 335 n.d. (29) II-30

 211 ± 67 n.d. (6.1) II-31

 962 ± 159 n.d. (64) II-32

2560 ± 442 n.d. (44) ^(a)Data are expressed as means ± SD of singledeterminations obtained in triplicate. ^(b)n.d. = not detected. ^(c)The% inhibition of the compound at a concentration of 10 μM against mPGES-1(IC₅₀ values were determined if the compounds resulted in 70% or higherinhibition).

Example 9 Characterization of Inhibition of Compounds of Formulae IIIand IV

TABLE 3 IC₅₀ ^(a) against mPGES-1 (nM) Compound Structure Human enzymeMouse enzyme III-01

1920 ± 300 n.d. (48) III-02

 613 ± 103 2880 ± 496 III-03

n.d.^(b) (63)^(c) n.d. (55)  IV-01

_ 296 ± 48 n.d. (91) ^(a)Data are expressed as means ± SD of singledeterminations obtained in triplicate. ^(b)n.d. = not detected. ^(c)The% inhibition of the compound at a concentration of 10 μM against mPGES-1(IC₅₀ values were determined if the compounds resulted in 70 % or higherinhibition).

Example 10 Characterization of Inhibition of Compounds of Formula V

TABLE 4 IC₅₀ ^(a) against mPGES-1 (nM) ID. Structure Human enzyme Mouseenzyme V-01

 8739 ± 1169 n.d.^(b) V-02

4817 ± 511 n.d. V-03

4749 ± 489 n.d. V-04

285 ± 40 754 ± 73  V-05

135 ± 16 776 ± 217 V-06

 89 ± 12 716 ± 120 V-07

6225 ± 502 n.d. V-08

5241 ± 429 n.d. V-09

3518 ± 471 n.d. V-10

136 ± 13 1390 ± 255  V-11

376 ± 31 n.d. V-12

 998 ± 196 n.d. V-13

181 ± 33 1632 ± 250  V-14

1008 ± 262 n.d. V-15

 83 ± 14 357 ± 52  V-16

1297 ± 232 n.d. V-17

1865 ± 350 n.d. V-18

74 ± 8 572 ± 83  V-19

2270 ± 350 n.d. V-20

50 ± 9 270 ± 64  V-21

5633 ± 987 n.d. V-22

 348 ± 100 1771 ± 241  V-23

1448 ± 192 n.d. V-24

 294 ± 060 n.d. V-25

242 ± 30 n.d. V-26

 905 ± 177 n.d. V-27

 51 ± 10 390 ± 84  V-28

4374 ± 915 n.d. V-29

2880 ± 687 n.d. V-30

1095 ± 212 n.d. V-31

 356 ± 123 n.d. V-32

 4283 ± 1404 n.d. V-33

2210 ± 450 n.d. V-34

 531 ± 116 n.d. V-35

256 ± 33 7291 ± 2546 V-36

541 ± 82 n .d. V-37

5414 ± 818 n.d. V-38

10811 ± 1038 n.d. V-39

1451 ± 152 n.d. V-40

1455 ± 119 n.d. V-41

4140 ± 858 n.d. V-42

 439 ± 104 n.d. V-43

2772 ± 577 n.d. V-44

 456 ± 042 n.d. ^(a)Data are expressed as means ± SD of singledeterminations obtained in triplicate. ^(b)n.d. = not detected.^(c)Known compounds were labeled with CAS

Example 11 Characterization of Inhibition of Compounds of Formulae VIand VII

TABLE 5 IC₅₀ ^(a) against mPGES-1 (nM) Name Structure Human Mouse  VI-01

265 ± 96 n.d.^(b) (28)^(c)  VI-02

212 ± 34 2573 ± 628  VI-03

169 ± 41 357 ± 75  VI-04

285 ± 65 n.d. (46)  VI-05

323 ± 52 2157 ± 188  VI-06

361 ± 51  740 ± 108  VI-07

 375 ± 127 n.d. (21)  VI-08

294 ± 83 n.d.(24)  VI-09

 598 ± 142 n.d. (0)  VI-10

 95 ± 16 n.d. (46)  VI-11

 92 ± 20 1264 ± 138  VI-12

 56 ± 10 445 ± 83  VI-13

 52 ± 15  1769 ± 1158  VI-14

113 ± 23 1126 ± 131  VI-15

 92 ± 19 316 ± 30  VI-16

188 ± 31 n.d. (50)  VI-17

 93 ± 14 n.d. (56)  VI-18

 797 ± 160 n.d. (25)  VI-19

n.d. (30) n.d.  VI-20

n.d. (29) n.d.  VI-21

n.d. (16) n.d.  VI-22

n.d. (6.8) n.d.  VI-23

n.d. (0) n.d.  VI-24

n.d. (0) n.d. VII-01

n.d. (32) n.d. VII-02

n.d. (14) n.d. VII-03

n.d. (24) n.d. VII-04

n.d. (37) n.d. VII-05

1593 ± 557 n.d. VII-06

1394 ± 303 n.d. VII-07

 4932 ± 1161 n.d. VII-08

1036 ± 293 n.d. VII-09

1729 ± 666 n.d. VII-10

41 ± 5 n.d. (35) VII-11

 36 ± 11 n.d. (37) VII-12

282 ± 83 n.d. (12) VII-13

296 ± 68 n.d. (21) VII-14

190 ± 68 n.d. (30) VII-15

 83 ± 30 n.d. (48) VII-16

209 ± 42 n.d. (46) VII-17

 90 ± 15 n.d. (29) VII-18

197 ± 32 n.d. (4.3) VII-19

 97 ± 15 n.d. (4.6) VII-20

 806 ± 162 n.d. (16) VII-21

n.d. (51) n.d. VII-22

n.d. (54) n.d. VII-23

n.d. (77) n.d. VII-24

n.d. (64) n.d. VII-25

n.d. (55) n.d. VII-26

n.d. (52) n.d. VII-27

n.d. (49) n.d. VII-28

n.d. (47) n.d. ^(a)Data are expressed as means ± SD of singledeterminations obtained in triplicate. ^(b)n.d. = not detected. ^(c)The% inhibition of the compound at a concentration of 10 μM againstmPGES-1.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, the definitions set forth herein are providedto facilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “an inhibitor” includes aplurality of such inhibitors, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±50%, in someembodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, insome embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%,in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed method.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Throughout this document, various references are mentioned. All suchreferences, including those listed below, are incorporated herein byreference.

REFERENCES

-   1. Serhan, C. N.; Levy, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,    8609.-   2. Kudo, I.; Murakami, M. J. Biochem. Mol. Biol. 2005, 38, 633.-   3. Fahmi, H. Current Opinion in Rheumatology 2004, 16, 623.-   4. Park, J. Y; Pillinger, M. H.; Abramson, S. B. Clinical Immunology    2006, 119, 229.-   5. Murakami, M.; Nakatani, Y; Tanioka, T.; Kudo, I. Prostaglandins    Other Lipid Mediators 2002, 68-9, 383.-   6. Murakami, M.; Naraba, H.; Tanioka, T.; Semmyo, N.; Nakatani, Y;    Kojima, F.; Ikeda, T.; Fueki, M.; Ueno, A.; Oh-ishi, S.; Kudo, I. J.    Biol. Chem. 2000, 275, 32783.-   7. Uematsu, S.; Matsumoto, M.; Takeda, K.; Akira, S. Journal of    Immunology 2002, 168, 5811.-   8. Kamei, D.; Murakami, M.; Nakatani, Y; Ishikawa, Y; Ishii, T.;    Kudo, I. Journal of Biological Chemistry 2003, 278, 19396.-   9. Kamei, D.; Yamakawa, K.; Takegoshi, Y; Mikami-Nakanishi, M.;    Nakatani, Y; Oh-ishi, S.; Yasui, H.; Azuma, Y; Hirasawa, N.; Ohuchi,    K.; Kawaguchi, H.; Ishikawa, Y; Ishii, T.; Uematsu, S.; Akira, S.;    Murakami, M.; Kudo, I. Journal of Biological Chemistry 2004, 279,    33684.-   10. Ikeda-Matsuo, Y; Ota, A.; Fukada, T.; Uematsu, S.; Akira, S.;    Sasaki, Y. Proceedings of the National Academy of Sciences of the    United States of America 2006, 103, 11790.-   11. Murakami, M.; Kudo, I. Progress in Lipid Research 2004, 43, 3.-   12. Claveau, D.; Sirinyan, M.; Guay, J.; Gordon, R.; Chan, C. C.;    Bureau, Y; Riendeau, D.; Mancini, J. A. Journal of Immunology 2003,    170, 4738.-   13. Oshima, H.; Oshima, M.; Inaba, K.; Taketo, M. M. EMBO Journal    2004, 23, 1669.-   14. Friesen, R. W.; Mancini, J. A. Journal of Medicinal Chemistry    2008, 51, 4059.-   15. Samuelsson, B.; Morgenstern, R.; Jakobsson, P. J.    Pharmacological Reviews 2007, 59, 207.-   16. Scholich, K.; Geisslinger, G. Trends in Pharmacological Sciences    2006, 27, 399.-   17. Cheng, Y; Wang, M.; Yu, Y; Lawson, J.; Funk, C. D.;    FitzGerald, G. A. Journal of Clinical Investigation 2006, 116, 1391.-   18. Engblom, D.; Saha, S.; Engstrom, L.; Westman, M.; Audoly, L. P.;    Jakobsson, P. J.; Blomqvist, A. Nature Neuroscience 2003, 6, 1137.-   19. Trebino, C. E.; Stock, J. L.; Gibbons, C. P.; Naiman, B. M.;    Wachtmann, T. S.; Umland, J. P.; Pandher, K.; Lapointe, J. M.; Saha,    S.; Roach, M. L.; Carter, D.; Thomas, N. A.; Durtschi, B. A.;    McNeish, J. D.; Hambor, J. E.; Jakobsson, P. J.; Carty, T. J.;    Perez, J. R.; Audoly, L. P. Proceedings of the National Academy of    Sciences of the United States of America 2003, 100, 9044.-   20. Thoren, S.; Jakobsson, P. J. Eur. J. Biochem. 2000, 267, 6428.-   21. Mancini, J. A.; Blood, K.; Guay, J.; Gordon, R.; Claveau, D.;    Chan, C. C.; Riendeau, D. J. Biol. Chem. 2001, 276, 4469.-   22. Riendeau, D.; Aspiotis, R.; Ethier, D.; Gareau, Y.; Grimm, E.    L.; Guay, J.; Guiral, S.; Juteau, H.; Mancini, J. A.; Methot, N.;    Rubin, J.; Friesen, R. W. Bioorg. Med. Chem. Lett. 2005, 15, 3352.-   23. De Simone, R.; Chini, M. G.; Bruno, I.; Riccio, R.; Mueller, D.;    Werz, O.; Bifulco, G. Journal of Medicinal Chemistry 2011, 54, 1565.-   24. Quraishi, O.; Mancini, J. A.; Riendeau, D. Biochem. Pharmacol.    2002, 63, 1183.-   25. Lu, J.; Wu, L.; Jiang, J.; Zhang, X., Helical Nanostructures of    an Optically Active Metal-Free Porphyrin with Four Optically Active    Binaphthyl Moieties: Effect of Metal-Ligand Coordination on the    Morphology. European Journal of Inorganic Chemistry 2010, 2010 (25),    4000-4008.-   26. Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.;    Engelhardt, E. L., Cyclization-activated prodrugs. Basic carbamates    of 4-hydroxyanisole. Journal of medicinal chemistry 1990, 33 (1),    97-101.-   27. Baron, R.; Huang, C. H.; Bassani, D. M.; Onopriyenko, A.;    Zayats, M.; Willner, I., Hydrogen-bonded CdS nanoparticle assemblies    on electrodes for photoelectrochemical applications. Angewandte    Chemie 2005, 44 (26), 4010-5.-   28. Hidalgo-Figueroa, S.; Ramirez-Espinosa, J. J.; Estrada-Soto, S.;    Almanza-Perez, J. C.; Roman-Ramos, R.; Alarcon-Aguilar, F. J.;    Hernandez-Rosado, J. V.; Moreno-Diaz, H.; Diaz-Coutino, D.;    Navarrete-Vazquez, G., Discovery of    thiazolidine-2,4-dione/biphenylcarbonitrile hybrid as dual PPAR    alpha/gamma modulator with antidiabetic effect: in vitro, in silico    and in vivo approaches. Chemical biology & drug design 2013, 81 (4),    474-83.-   29. Yan, Q.; Cao, R.; Yi, W.; Chen, Z.; Wen, H.; Ma, L.; Song, H.,    Inhibitory effects of 5-benzylidene barbiturate derivatives on    mushroom tyrosinase and their antibacterial activities. European    journal of medicinal chemistry 2009, 44 (10), 4235-43.-   30. Komiya, M.; Asano, S.; Koike, N.; Koga, E.; Igarashi, J.;    Nakatani, S.; Isobe, Y., Structure and activity relationship of    2-(substituted benzoyl)-hydroxyindoles as novel CaMKII inhibitors.    Bioorganic & medicinal chemistry letters 2011, 21 (5), 1456-8.-   31. Chen, H.; Tsalkova, T.; Chepurny, O. G.; Mei, F. C.; Holz, G.    G.; Cheng, X.; Zhou, J., Identification and characterization of    small molecules as potent and specific EPAC2 antagonists. Journal of    medicinal chemistry 2013, 56 (3), 952-62.-   32. Murugan, R.; Anbazhagan, S.; Lingeshwaran; Sriman Narayanan, S.,    Synthesis and in vivo antidiabetic activity of novel    dispiropyrrolidines through [3+2] cycloaddition reactions with    thiazolidinedione and rhodanine derivatives. European journal of    medicinal chemistry 2009, 44 (8), 3272-9.-   33. Shibinskaya, M. O.; Lyakhov, S. A.; Mazepa, A. V.; Andronati, S.    A.; Turov, A. V.; Zholobak, N. M.; Spivak, N. Y., Synthesis,    cytotoxicity, antiviral activity and interferon inducing ability of    6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines. European journal of    medicinal chemistry 2010, 45 (3), 1237-43.-   34. Yamamoto, Y.; Yohda, M.; Shirai, T.; Ito, H.; Miyaura, N.,    Me-BIPAM for the synthesis of optically active    3-aryl-3-hydroxy-2-oxindoles by ruthenium-catalyzed addition of    arylboronic acids to isatins. Chemistry, an Asian journal 2012, 7    (10), 2446-9;-   35. Kaila, N.; Janz, K.; Huang, A.; Moretto, A.; DeBernardo, S.;    Bedard, P. W.; Tam, S.; Clerin, V.; Keith, J. C., Jr.; Tsao, D. H.;    Sushkova, N.; Shaw, G. D.; Camphausen, R. T.; Schaub, R. G.; Wang,    Q.,    2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylic    acid (PSI-697): identification of a clinical candidate from the    quinoline salicylic acid series of P-selectin antagonists. Journal    of medicinal chemistry 2007, 50 (1), 40-64.-   36. Yamazaki, K.; Kaneko, Y.; Suwa, K.; Ebara, S.; Nakazawa, K.;    Yasuno, K., Synthesis of potent and selective inhibitors of Candida    albicans N-myristoyltransferase based on the benzothiazole    structure. Bioorganic & medicinal chemistry 2005, 13 (7), 2509-22.-   37. Xie, J.; Seto, C. T., A two stage click-based library of protein    tyrosine phosphatase inhibitors. Bioorganic & medicinal chemistry    2007, 15 (1), 458-73.-   38. Cumpstey, I.; Carlsson, S.; Leffler, H.; Nilsson, U. J.,    Synthesis of a phenyl thio-beta-D-galactopyranoside library from    1,5-difluoro-2,4-dinitrobenzene: discovery of efficient and    selective monosaccharide inhibitors of galectin-7. Organic &    biomolecular chemistry 2005, 3 (10), 1922-32.-   39. Shete, D. K., et al., Comparative efficiency of metal phosphates    as a protomer in multi-component condensation reaction. Pharm.    Lett., 2010. 2(3): p. 59-65.-   40. de Vasconcelos, A., et al., Antioxidant capacity and    environmentally friendly synthesis of dihydro-(2H)-pyrimidinones    promoted by naturally occurring organic acids. J. Biochem. Mol.    Toxicol., 2012. 26(4): p. 155-161.-   41. Rathelot, P., et al., 1,3-Diphenylpyrazoles: synthesis and    antiparasitic activities of azomethine derivatives. European Journal    of Medicinal Chemistry, 2002. 37(8): p. 671-679.-   42. Stella, A., et al., Synthesis of a 2,4,6-trisubstituted    5-cyano-pyrimidine library and evaluation of its immunosuppressive    activity in a Mixed Lymphocyte Reaction assay. Bioorg. Med.    Chem., 2013. 21(5): p. 1209-1218.

1. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein R¹ is selectedfrom the group consisting of H, halide, Me, OMe, OEt, NO₂, OH, and,together with the ring to which it is attached, a bicyclic ring system;wherein R² is alkyl; wherein R³ is selected from the group consisting ofH and Me; and wherein X is selected from the group consisting of O or S;or

or pharmaceutically acceptable salts thereof, wherein R1 is selectedfrom the group consisting of H, an alkyl, an alkyl halide, an ether, anda carboxylic acid; wherein R2 is selected from the group consisting ofH, a halide, an alkyne, and an aromatic; and wherein R3 is selected fromthe group consisting of H, a carboxyl, a carboxylic acid, and an alkyl;or

or pharmaceutically acceptable salts thereof; wherein R is selected fromthe group consisting of an alkyl and an alkoxy; and wherein n is from 1to 6; or

or pharmaceutically acceptable salts thereof; wherein R is a substitutedphenyl; and wherein X is O; or

or pharmaceutically acceptable salts thereof; wherein R is selected fromthe group consisting of an aliphatic side chain and an alkyl; wherein Xis selected from the group consisting of H, NO₂, Br, and OMe; andwherein R′ and R″ are independently selected from the group consistingof CN, COOH, COOEt, CONH₂, and NO₂; or

or pharmaceutically acceptable salts thereof, wherein R¹ is an alkyl;wherein each R² is independently selected from the group consisting of Hand an alkyl; wherein X is selected from the group consisting of H and ahalogen; and wherein Y is selected from the group consisting of S and O;or

or pharmaceutically acceptable salts thereof; wherein R is selected fromthe group consisting of an aliphatic side chain and an alkyl; wherein Xis selected from the group consisting of H and Cl; wherein Y is CN;wherein Z is selected from the group consisting of CN, COOH, and,together with Y, a heterocyclic group of the formula:

wherein R¹ is selected from the group consisting of O and S; and whereinR² is selected from the group consisting of H and CH₂COOH.
 2. Thecompound of claim 1, wherein R¹ is selected from the group consistingof: H, Cl, Br, I, Me, OMe, OEt, NO₂, OH, and, taken together with thering to which it is attached, a bicyclic ring system; and/or wherein R²is selected from the group consisting of:


3. (canceled)
 4. The compound of claim 1, having the formula selectedfrom the group consisting of:

5-6. (canceled)
 7. The compound of claim 1, wherein R¹ is selected fromthe group consisting of: H,

and/or R² is selected from the group consisting of: H,

and/or wherein R³ is selected from the group consisting of: H

8-9. (canceled)
 10. The compound of claim 1, having the formula selectedfrom the group consisting of:


11. (canceled)
 12. The compound of claim 1, wherein R is selected fromthe group consisting of:


13. The compound of claim 1, having the formula selected from the groupconsisting of:


14. (canceled)
 15. The compound of claim 1, having the formula


16. (canceled)
 17. The compound of claim 1, having the formula selectedfrom the group consisting of:


18. (canceled)
 19. The compound of claim 144, having the formulaselected from the group consisting of


20. (canceled)
 21. The compound of claim 1, having the formula selectedfrom the group consisting of:


22. A pharmaceutical composition, comprising a compound of claim 1 and apharmaceutically-acceptable carrier.
 23. The pharmaceutical compositionof claim 22, further comprising: a second compound or composition havingmPGES-1 inhibition activity, having anti-inflammatory activity, beinguseful for treatment of an inflammation disorder, being useful fortreatment of symptoms associated inflammation and/or an inflammationdisorder, or combinations thereof. 24-25. (canceled)
 26. A method ofreducing inflammation in a subject, comprising administering to thesubject an effective amount of a compound of claim
 1. 27. The method ofclaim 26, wherein the subject includes an inflammation disorder orsymptoms thereof.
 28. The method of claim 27, wherein the inflammationdisorder is selected from the group consisting of inflammation,arthritis, fever, pain, cancer, stroke, bone disorders, and combinationsthereof.
 29. The method of claim 26, wherein the compound inhibitsmicrosomal prostaglandin E synthase-1 (mPGES-1).
 30. A method ofreducing inflammation in a subject, comprising administering to thesubject an effective amount of a compound selected from the groupconsisting of:


31. The method of claim 30, wherein the subject includes an inflammationdisorder or symptoms thereof.
 32. (canceled)
 33. The method of claim 30,wherein the compound inhibits microsomal prostaglandin E synthase-1(mPGES-1).